Administration of many chemical agents results in induction of malignant tumors in target tissues. These malignancies are characterized by alterations in control of cellular function, suggesting that genomic alteration (mutation) and/or alterations in gene modulation have occurred. While a substantial body of evidence indicates that altered DNA plays a major role in this process, the role of non-DNA factors has also been demonstrated. In this continuation, we will extend our two-dimensional gel electrophoretic study of potentially critical alterations occurring in nonhistone chromosomal proteins (NHCP) and cytosolic proteins during carcinogenesis induced by diverse acting carcinogens acetylaminofluorene (AAF) and diethylnitrosamine (DEN), and in the cancers that result. We have demonstrated that the gel system provides a highly consistent pattern of protein separation, with extreme resolution, over long periods of time. Silver and in vitro radiolabeling techniques have been developed to permit visualization of nanogram amounts of protein(s) with applicability to computer technology. Using these techniques, we have identified induction of tumor-associated proteins (TAP) in the total NHCP fraction as well as in the phenol-soluble fraction (phosphoproteins) of rat hepatic chromatins during AAF and DEN carcinogenesis and resulting hepatocellular carcinomas. Additionally, cytosolic AAF and DEN-TAPs have been similarly demonstrated. We will determine to what extent these changes in NHCP and cytosolic proteins characterize the general process of carcinogenesis by examining spontaneous and chemically-induced hepatic and thymus tumors in a variety of mouse inbred strains. We will further determine the time of appearance, sub-fraction location, and in the case of NHCP, genomic distribution of TAP during chemical carcinogenesis; isolate these proteins, and produce monoclonal antibodies for use in their study, as well as determining their functional significance. Lastly, we will continue our technological developments to assure maximum detection, and computer-assisted image-processing of the complex protein patterns that characterize normal and malignant cells in order to confirm and further detect potential hallmark proteins, whose occurrence mark malignant induction.